Comparison of assimilation efficiency on diets of nine phytoplankton species of the greenshell mussel Perna canaliculus.

ABSTRACT The greenshell mussel Perna canaliculus is the most important species in aquaculture in New Zealand. Mussel energetics and growth rates are subject to the natural variability in phytoplankton biomass and species composition and thus understanding the influence of food type on assimilation efficiency is fundamental to the prediction of mussel production and planning farm management. In this study pulse-chase feeding techniques were used to assess the effect of diet on assimilation efficiencies for nine phytoplankton species including three diatoms Chaetoceros calcitrans, Skeletonema costatum and Thalassiosira sp., and three flagellates Eutreptiella sp., Pyramimonas sp. and Isochrysis galbana, and three dinoflagellates Akashiwo sanguineum, Alexandrium minutum and Gymnodinium catenatum. Assimilation efficiency varied with algal species, but it was significantly higher when mussels were fed dinoflagellates (84.5%) compared with diatoms (61.7%) and flagellates (77.9%). Assimilation efficiency of dinoflagellates and flagellates increased with gut passage time, whereas with a diatom diet, a negative correlation was evident. This finding has implications in understanding and predicting growth rates of mussels (and hence commercial yield) in tandem with natural variability in phytoplankton species composition.

KEY WORDS: mussel, Perna canaliculus, phytoplankton, assimilation efficiency, gut passage time

INTRODUCTION

The feeding responses of bivalves to variations in the quantity and quality of seston have been extensively studied (see reviews by Winter 1978 and Jorgensen 1996). Results of these studies have shown the ability of bivalves to preferentially ingest organic particles from the filtered matter and selectively reject the inorganic particles. Many of these feeding experiments have been conducted on blue mussels (e.g., Bayne et al. 1989, Bayne et al. 1993, Navarro et al. 1992), oysters (e.g., Newell & Jordan 1983, Barille et al. 1997, Ren et al. 2000), scallops (e.g., Cranford et al. 1998), clams (e.g., Hartwell et al. 1991), and cockles (Iglesias et al. 1992 & Iglesias et al. 1996). Few studies used P. canaliculus, the greensbell mussel (Hawkins et al. 1999). In these studies, diets of either natural seston or natural food with addition of algae were used to understand the effects of food quantity and quality on feeding dynamics. Seston-phytoplankton mixed diets generate a blended assimilation that reflects a diversity of particle sources and quality. To our knowledge however, relatively little attention has been paid to the preferential ingestion among phytoplankton species (e.g., Romberger & Epifanio 1981, Shumway et al. 1985).

Considerable intra-annual variations in growth rates and conditions of farmed shellfishes have been observed in Marlborough Sounds, New Zealand (e.g., Ross et al. 1997, Ross & Image 2001). This reflects the complex ecophysiological response of mussels to the equal complexity of interactions among environmental variables. High phytoplankton biomass usually results in fast growth and increase in condition of mussels (Ren & Ross 2005). Chlorophyll-a pigment concentration is often used to estimate phytoplankton biomass in field monitoring and, therefore, can be an indicator of environmental conditions that influence mussel growth. Over the last few years, the chlorophyll-a concentration in Marlborough Sounds has generally decreased and there have been concurrent decreases in both the condition and growth rate of the mussels (e.g., Ross et al. 1997, Ross & Image 2001). Although these trends appear to be broadly related, there have been occasions when mussel growth has been poor even though surrounding chlorophyll-a levels have been moderate (Ren & Ross 2002). These discrepancies may relate to the differential consumption of phytoplankton species or groups. During the same period, the composition and abundance of the phytoplankton was characterized by strong intra and interannual variations in the extensive mussel farming area of Marlborough Sounds. This variation may even be pronounced during seasons of algal blooms when one single species usually dominates the phytoplankton biomass (Mackenzie et al. 1986, Ogilvie 2000). This raises the question on whether mussels can assimilate some algal species with greater efficiency than others and hereby show good growth even when chlorophyll-a as an indicator of total phytoplankton biomass is low. Selective ingestion on different algal species has been observed in other mussel species (e.g., Kreeger et al. 1996, Wang & Fisher 1996). The mussel Mytilus galloprovincialis preferentially selects dinoflagellates rather than diatoms and digestion rate of the dinoflagellates may also vary among species (Sidari et al. 1998). Although a few studies on feeding behavior of P. canaliculus have been done (Hawkins et al. 1999), very little is known of the selective assimilation in this species. To assess energy flows through a mussel-farmed ecosystem it is essential to quantify the efficiency with which the mussels assimilate the phytoplankton species and groups that inhabit the Marlborough Sounds.

Assimilation efficiency is usually estimated from direct measurements based on the ash ratio between the food source and feces as developed by Conover (1966). This ratio is embodied in the mass balance equation that assesses scope for growth, which is based on measured difference between energy gains from feeding and energy loss for respiration and excretion (Baynes & Newell 1983). However, the relationship between estimated growth from mass balance models and actual growth has not been convincingly described in bivalves (e.g., Riisgard & Randol 1981). By comparing estimated and actual growth of mussels, the authors found that estimation of assimilation efficiency gave unreliable results. Although uncertainty in modeling techniques may not be ruled out (see van Haren & Kooijman 1993), limitations of the methods used in physiological measurements may, to some extent, have contributed to the error of estimated assimilation efficiency. As discussed by Conover (1966), when the diet consists of a low ratio of ash to organic content such as phytoplankton, a significant proportion of ash can be absorbed. This would result in uncertainties in estimated assimilation under such a condition. This method also requires a large amount of material and is susceptible to weighing errors even when using a microbalance (Navarro & Thompson 1994). In shellfish farming ecosystems such as Marlborough Sounds of New Zealand, the contribution of phytoplankton biomass to total organic particulate matter is usually very low in terms of dry weight, but it is the main energy sources for bivalve growth. Small errors in measurements can potentially result in considerable larger errors in estimation of energy budgets (Ren & Ross 2005). Therefore, accuracy in estimates of the bioenergetic parameter is critical for the study of mussel energetics.

In this study, we used a radiotracer technique to investigate how mussels respond to different food diets. Assimilation efficiencies were measured in P. canaliculus using nine phytoplankton species found in a mussel-farming ecosystem in Marlborough Sounds, New Zealand. This study is aimed at providing information for parameterizations of energetic and carrying capacity models currently being developed (Ren & Ross 2005).

MATERIALS AND METHODS

Mussels

Three hundred mussels (P. canaliculus, ~70 cm shell length) were collected on one occasion from a farming site in Marlborough Sounds, New Zealand. They were transported immediately to and kept on ropes in Magazine Bay near Christchurch for use in the experiments. For the feeding trial, 54 mussels were transferred to the NIWA laboratory in Christchurch and acclimatized in a recirculating aerated flow-through seawater system for 72 h prior to feeding experiments. During the acclimatization period, mussels were fed with a diet of mixed algae consisting of nine experimental algae with equal numbers of each species. Approximately 1 x [10.sup.9] cells of the algae were added each day per mussel to ensure that physiological activities of the animals were not affected because of starvation. Seawater was collected directly from Christchurch coast and stored in three holding tanks. Because the seawater was kept in the dark and aerated in the laboratory for over 6 wks before experiments, there were no living algae in the water at the time of use. Seston, particulate organic matter and chlorophyll-a concentrations were 2.15 mg [L.sup.-l], 0.49 mg [L.sup.-1] and ~0 [micro]g [L.sup.-1] respectively. Temperature was constant at ~16[degrees]C. The seawater was recirculated and monitored once a day for ammonia concentration using ammonia test kits. Throughout all experiments, the ammonia concentration never exceeded 1 ppm.

Experimental Diets

An important aspect of this study was to measure assimilation efficiency that can be incorporated into energetic and carrying capacity models applied in mussel farming ecosystems. It is therefore important that these measurements be conducted on diets reflecting natural conditions. In mussel farming sites of the Marlborough Sounds, there is little variation in POM, whereas there is a considerable inter and intra annual variation in phytoplankton species composition. Phytoplankton contributes very little to POM in terms of dry weight (<10%, e.g., Ren 2001), but it is the major food sources for bivalve growth in terms of available suspended particulates (e.g., James & Ross 1996, Ogilvie 2000). Therefore, to be representative of the natural condition, experimental diets were based on [sup.14]C labeled algae.

Nine phytoplankton species, Chaetoceros calcitrans, Skeletonema costatum, Thalassiosira sp., Akashiwo sanguineum, Alexandrium minutum, Gymnodinium catenatum, Eutreptiella sp., Isochrysis galbana, Pyramimonas sp., belonged to diatom, flagellate and dinoflagellate, were cultured at the NIWA aquaculture laboratory at Mahanga Bay. The algae were enumerated by particle counter (Model ZM and tube with 100-[micro]m orifice diameter). The algae were spiked with [sup.14]C (100 [micro]Cu [mL.sup.-1]) by exposing the algae to the radiotracers for 3 d and kept in 12:12 light-dark photoperiod at 17[degrees]C. To calculate [sup.14]C-uptake after 3 d exposure, 2 mL replicate samples were each filtered through a 25 mm diameter GF/F filters (0.7-[micro]m pore, Whatman). These filters were then added to 1 ml 5% HCL and held for over 6 h for liquid scintillation cocktail for analysis (LKB Wallac 1217 Rackbeta) of [sup.14]C-activity. Before feeding to mussels, the algal cells were separated from their culture medium by centrifugation (1000 g, 10 min) to remove dissolved [sup.14]C. The algal pellets were rinsed with 0.7-[micro]m filtered seawater. This process was repeated three-times to remove surface attached chemicals. The cells were resuspended in 100 mL seawater and enumerated for the feeding experiments as described below.

Experiments with Mussels

Radiolabeled algae were transferred into a 20-L container and mixed with 15 L seawater (no living algae). Nine experimental trials were conducted simultaneously and experimental diets were prepared by adding [sup.14]C labeled algae into seawater. Each experimental trial was identical except that mussels were fed on different [sup.14]C labeled algal species. Six replicate individual mussels were then placed in the container containing the mixture of seawater and radiolabeled algae (~1 x [10.sup.5] cells [mL.sup.-1]) for 30 min. The duration of the feeding was assumed sufficient to allow mussels to ingest a substantial amount of the labeled algae but not defecate, because the experimental duration was less than gut transition time of 80 min (Hawkins et al. 1999). The solution was stirred and aerated to homogenize algae during the course of the feeding trial. Pseudofeces production was not observed; this was probably caused by particulate concentrations of the diets being lower than the threshold for pseudofeces production. Feces were collected at the end of the feeding trial, but [sup.14]C activity in these were negligible.

After feeding with radiolabeled algae, the mussels were rinsed with seawater and transferred into a flow-through system to "chase" the unassimilated material through the gut. The mussels were fed with unlabeled mixed algal diet the same as prior to the experiment. In a separate pilot trial, gut evacuation time of the mussel varied with algal species, but most of ingested diets were evacuated within 30 h. In this experiment, mussels were allowed to evacuate their guts for 3 d. This was also based on evidence that Mytilus edulis completes its digestion and assimilation of food within 3 d (see Wang et al. 1995). Feces produced by individual mussels were collected with a wide-pore pipette to monitor gut passage of the radioactive algae every 1-3 h for the first 10 h and every 4-20 h thereafter. Feces were filtered onto 25 mm diameter GF/F filters (0.7 [micro]m pore, Whatman) and individually placed into 20 mL scintillation vials until analysis.

After a 3 d pulse-chase feeding experiment, ingested [sup.14]C algae were to have been assimilated and incorporated in the mussel flesh, which was then dissected from the shell, added to a tared aluminum weighing pan, freeze-dried and then reweighed to determine total dry flesh weight of each mussel. Dried flesh of individual mussels was then ground with a mortar and pestle and three replicates of ~200 mg were placed into 20 mL scintillation vials for counts of incorporated [sup.14]C. The powdered sample was homogenized in 1 mL of distilled water and left overnight at room temperature. The next day, 1 mL of tissue solubilizer was added into each vial, which were then incubated in a water bath at 95[degrees]C for 5 h before 10 mL "HiSafe"-III liquid scintillation cocktail was added. The homogenate solution was thoroughly mixed, left overnight and counted using a liquid scintillation counter (LSC) to determine [sup.14]C incorporation. Resulting counts per minute were converted to decays per minute using a quench curve and the external standard technique with correction for background radioactivity.

For the analysis of [sup.14]C in feces, 1 mL of 5% HCL was added into each vial containing feces and left overnight to breakdown any possible undigested algal cells. Ten milliliters "HiSafe"-III liquid scintillation cocktail was added into each vial and [sup.14]C was counted using LSC as above. The total [sup.14]C activity was summarized from all collections of feces during the 3 d pulse-chase feeding period.

Statistical Analysis

Assimilation efficiency (AE) was defined as the proportion of ingested [sup.14]C retained after completion of digestion and gut evacuation of [sup.14]C within 3 d, as

AE = [sup.14][C.sub.flesh]/([sup.14][C.sub.flesh] + [sup.14][C.sub.feces])

where [sup.14][C.sub.flesh] is [sup.14]C incorporated in the flesh and [sup.14][C.sub.feces] is [sup.14]C egested in the feces during the 72 h pulse-chase feeding period. This method is based on the assumption that respired and excreted [sup.14]C are negligible (e.g., Wang & Fisher 1996, Charles & Newell 1997). For example, Charles & Newell (1997) showed that respired [sup.14]C contributed as little as 0.4% to the total ingested [sup.14]C in the mussel Geukensia demissa.

Following Wang & Fisher (1996), gut passage time (GPT) is defined as the time at which 90% of the accumulated [sup.14]C in the feces is recovered, assuming 100% recovery at 3 d pulse-chase feeding period.

Multiple comparison tests on data were used to detect significant differences of assimilation efficiency between food algal types (95% confidence level). Linear regression model was used to analyze correlation between AE and GPT. All analyses were done using S-PLUS, (Insightful Corp.)

RESULTS

Defecation and Gut Passage Time

No pseudofeces were detected during the experiments indicating that the concentration of total particulate matter was below the threshold for pseudofeces production. The defecation of nine-tested phytoplankton species in mussels after radioactive pulse feeding is shown in Figure 1. In general, the unassimilated portion of ingested radioactive phytoplankton showed a rapid loss through feces within the first few hours. However, the defecation rate varied considerably among phytoplankton species, being the fastest on a diet of Alexandrium minutum, Chaetoceros calcitrans and Skeletonema costatum, whereas Thalassiosira sp. and Gymnodinium catenatum were egested with slow rates. This defecation pattern can be shown more clearly using gut passage time (GPT) because this showed a large variation between food types, ranging from 6.9-50.9 h (Fig. 2). Despite variation in GPT, species-specific differences were not significant (P > 0.51). No significant differences in GPT were detected when the phytoplankton species were grouped into diatom, flagellate and dinoflagellate taxa (P > 0.71).

[FIGURES 1-2 OMITTED]

Assimilation Efficiency

Assimilation efficiencies of the nine-phytoplankton species are shown in Figure 3. The greenshell mussel assimilated phytoplankton with overall high rates, but average assimilation efficiency (AE) varied considerably with phytoplankton species, ranging from 55.6% to 87.7%. Overall, AEs of dinoflagellates were highest, whereas AEs of diatoms were lowest. The average efficiency with which the mussel assimilated the dinoflagellates (84.5 [+ or -] 6.3%) was significantly higher than that of diatoms (P < 0.01) (61.7 [+ or -] 11.0%). Similarly, mussels assimilated flagellates with significantly higher efficiency than diatoms (P < 0.01). However, the difference of AEs between flagellates (77.9 [+ or -] 6.7%) and dinoflagellates was not significant (P > 0.08). There were no significant differences of AE between species within diatom (P > 0.17), flagellate (P > 0.56) and dinoflagellate (P > 0.41).

[FIGURE 3 OMITTED]

Correlation analysis showed that AE of diatoms was negatively correlated to GPT ([R.sup.2] = -0.97, P > 0.11), but AEs of dinoflagellates and flagellates were positively correlated to GPT ([R.sup.2] = 0.1, P > 0.86). If Alexandrium minutum is excluded, this trend is more robust ([R.sup.2] = 0.49, P > 0.18).

DISCUSSION

The pulse-chase feeding technique was used in this study to specifically address the influence of phytoplankton species on assimilation efficiency in the greenshell mussel Perna canaliculus. We chose to use nine phytoplankton species to represent diatoms, dinoflagellates and flagellates that are found in the mussel farming areas in New Zealand. The results have demonstrated that AE varied between phytoplankton species, but AEs of dinoflagellates and flagellates was generally higher than those of diatoms.

The GPT in our study varied between 7 and 51 h, which was independent of algal species. Control of gut retention time has been demonstrated in Mytilus edulis and Cerastoderma edule and a strong positive relationship between absorption efficiency and gut retention time has been recorded in M. edulis (e.g., Hawkins et al. 1990, Wang & Fisher 1996, Chong & Wang 2000). Longer retention of ingested food within the digestive tract allows for more efficient digestion and assimilation (Willows 1992), and thus maximizes nutrient gain. Although this theory is supported by our experimental data that AEs of dinoflagellate and flagellate diets for which AE was positively correlated to GPT, AE of diatoms was negatively correlated to GPT. These results suggest that AE may not always increase with increasing GPT. Similar results have also been observed from two marine bivalves in which AE of pooled diatoms and flagellates was not correlated to GPT (Li et al. 2001). The assimilation efficiency of bivalves reflects complex physiological processes within an organism and can be influenced by many physiological factors including enzymatic digestive activity, gut residence time, gut capacity and digestive synchrony (e.g., Bayne & Newell 1983, Hawkins et al. 1990, Wang & Fisher 1996, Charles & Newell 1997). The adaptive strategies of bivalves in digestive processes (Bricelj et al. 1984) may help explain the differing results. A mussel would have little benefit in withholding relatively indigestible diatoms longer in the gut for more efficient digestion. The difference of diatom-AEs may have resulted from the variability in digestibility between the species. Relatively less digestible diatom species may be egested more rapidly. Conversely, the longer GPT of relatively digestible dinoflagellates and flagellates would have resulted in more efficiency.

Our observations demonstrated that mussels assimilated carbon from diatoms at a lower efficiency than they did from dinoflagellates and flagellates. This is probably typical for many bivalves, because the cell walls of diatoms are rigid and resistant to enzymatic digestion and physical breakdown, and diatoms also contains significantly higher quantities of inorganic matter. AEs measured in our experiments are comparable with previous studies in other bivalves. For example, Shumway et al. (1985) found that bivalve molluscs preferentially assimilated a crytomonad flagellate over a diatom. Variations in assimilation efficiency with different algal species have been observed in other marine bivalves (e.g., Romberger & Epifanio 1981, Wang & Fisher 1996). The assimilation of some algal species can be extremely low, which would result in low energy available for growth. For example, oysters Crassostrea virginica could hardly digest Platymonas suecica (Romberger & Epifanio 1981). Results from our experiments indicated that the greenshell mussel digested algal species with a relatively overall high AE (55.6% to 87.7%) compared with the mussel M. edulis (7% to 86%, Wang & Fisher 1996), the oyster C. virginica (6% to 74%, Romberger & Epifanio 1981).

Although assimilation of the greenshell mussel have been investigated, direct comparison of AE is difficult because previous studies used either different algal species (Hawkins et al. 1999) or natural seawater (Gardner 2000) or combination of single algal species and natural seawater (Hawkins et al. 1999). To our knowledge, only one study used the same algal species (Isochrysis) as the present study (Marsden & Weatherhead 1999). Our measured AE (76.3%) is lower than theirs (84%). Differences in experimental designs may explain the differing results. The previous study used intertidal mussels starved for 3-12 d before experimentation. The physiological behavior may have either differed from farmed mussels or have been affected by starvation or both. Moreover, their study used the Conover technique. This technique relies on measuring gravimetric changes in the ratio of organic material to ash between food and feces, which is susceptible to weighing errors (Navarro & Thompson 1994). Therefore, it is not as sensitive as the radiotracer technique that we used in this study. Because the radiotracer technique does not require a large amount of material, it is particularly accurate when food concentration is low and consists of a high proportion of algal particles.

Selective feeding on particles of different sizes has been reported in filter feeders (Newell & Jordan 1989, Barrille et al. 1993, Wang & Fisher 1996), but this selective behavior was only demonstrated in marine bivalves when fed with small particles. For example, experimental results by Barrille et al. (1993) showed no change in retention efficiency of oyster (Crassostrea gigas) for particles larger than 3-4 [micro]m. Wang & Fisher (1996) demonstrated that assimilation efficiency of M. edulis was not directly related to the particle size of different algae ranging from 2-40 [micro]m, which is in agreement with other studies (e.g., Newell & Jordan 1983). Similarly, the size of the algal cells in our study ranged from 4-38 [micro]m (data not showed) and was not significantly related to assimilation efficiency (P > 0.68). Therefore, algal sizes in this range may not be important during the digestive period and thus would not significantly affect assimilation efficiency.

We did not account for respiratory loss of [sup.14]C in the present study, which may have resulted in slight underestimates of AEs. However, the respiratory loss of [sup.14]C may be negligible according to similar experiments on other mussels (e.g., Kreeger 1993, Wang & Fisher 1996) and only 0.4% of ingested food went to C[O.sub.2] (Charles & Newell 1997). Although the contribution of respired [sup.14]C to AEs might be small, it is recommended that this term be included in the future measurements.

Studies on the food available to bivalve feeders in coastal waters reveal marked temporal variations in particulate matter concentration, composition and nutritional value (Bayne et al. 1993, Galois et al. 1996). Variability on a scale of days to weeks can result from episodic algal blooms and shifts in species composition. In the extensive mussel farming area of Marlborough Sounds, New Zealand, a high-frequency fluctuation of phytoplankton species composition has been observed (Mackenzie et al. 1986). For example, in early spring and summer of 1982 to 1983, micro-flagellates dominated the phytoplankton community (over 50%), whereas diatom species made up of less than 30% of the total biomass. In the autumn however, the diatoms dominated (>50%) (Mackenzie et al. 1986). An understanding of the influence of food type on assimilation efficiency is fundamental to validating models that predict not only greenshell mussel energetics and growth but also the capacity of the environment to support phytoplankton grazing by farmed mussels. Accurate models are essential for effective long-term farm management. There is a lack of comprehensive knowledge on assimilation efficiency and it is often assumed to be a constant or a functional response of ingested organic matter in many energetic modeling studies on farmed bivalves (Ren & Ross 2001, Ren & Ross 2005). An obvious and important aspect of the incorporation of AE into predictive models is obtaining values that are representative of the natural diet. Mussels have the ability to selectively ingest organic or high nutrient particles (e.g., Bayne & Hawkins 1990). If preferential assimilation of phytoplankton species has not been accounted for, then predictive models based on only one component of the diet such as organic content, may potentially under- or overestimate true energetics of the mussel. The present study has clearly demonstrated a considerable difference of physiological responses of the greenshell mussel to algal types.

It should be noted that AE might vary not only with phytoplankton species but also with different life stages of bivalves. For example, the diatom Skeletonema costatum is an excellent food for oyster juveniles (Walne 1970) but poor for larvae of the same species (Ostrea edulis) that favor flagellates (Enright et al. 1986, Ferreiro et al. 1990). Assimilation efficiencies of the phytoplankton species tested in our study are applicable to adult mussels. Further experiments are needed to investigate how juvenile and larval stages of the mussel respond to changes of food types.

In conclusion, the present study shows that assimilation efficiencies of adult P. canaliculus depend on the phytoplankton genera and/or species. This variability appeared to be related to differing digestibility of phytoplankton species caused by rigid cell walls of diatoms being more difficult for enzymatic digestion than dinoflagellates and flagellates. This finding would provide important information to the understanding of the physiological response of the mussel to the type of phytoplankton species. However, a diet of a single algal species may evoke a feeding behavior that differs from diets of multialgal species or natural seawater (e.g., Romberger & Epifanio 1981, Navarro et al. 1992, Hawkins et al. 1996). Before the results are incorporated into energetic and carrying capacity models, further experimental work should be conducted to investigate the mussel responses to mixed diets of multialgal species in field conditions.

ACKNOWLEDGMENTS

The authors thank Mark Weatherhead, Andrea Blackburn and Dave Rawlings for providing logistic supports for the experiment and Sealord Shellfish Ltd for providing mussel samples. Thanks to Magazine Bay Marina for holding the mussels. We gratefully acknowledge the valuable comments of Dr. Jeanie Stenton-Dozey and anonymous reviewers. This study was supported by the New Zealand Foundation of Research, Science and Technology, contract number C01X0507.

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JEFFREY S. REN, * ALEX H. ROSS AND BARBARA J. HAYDEN

National Institute of Water and Atmospheric Research, PO Box 8602, Christchurch, New Zealand

* Corresponding author. E-mail: [e-mail]